It was recently discovered that one of the immunogens associated with halothane-induced hepatitis is a trifluoroacetylated form of a 59 kDa liver microsomal carboxylesterase. In order to learn more about the structure, regulation, and physiological function of this enzyme and its role in halothane hepatitis, a study of its molecular biology has been initiated. Polylclonal anti-59 kDa antibodies raised in a rabbit was used to screen rat liver cDNA libraries constructed in the expression vector lambda gt11. Several positive clones were isolated, subcloned, and sequenced by double-stranded and shotgun cloning techniques in conjunction with dideoxy sequencing methodology. A peril sequence was assembled based upon these results and sequence information derived from tryptic peptides of the native 59 kDa protein that corresponded to approximately 2/3 of the projected length of functional 59 kDa protein. This sequence contained the putative active site regions found in other serine-type esterases. Further analysis of other clones is ongoing in order to yield complete protein and genetic data for the rat liver 59 kDa microsomal carboxylesterase and its possible isozymes. The clones will serve as genetic probes for the investigation of the tissue distribution and regulation of this enzyme and ultimately will help to elucidate its physiological function and role in halothane hepatitis.